Project Summary The hostile microenvironment of solid tumors is characterized by irregular vascularization and poor oxygen (O2) and nutrient supply. While normal cells adjust their rates of growth and proliferation in response to changes in nutrient availability by modulating anabolic and catabolic pathways, cancer cells exhibit unregulated growth even under nutrient scarcity. The mTORC1 complex is a particularly important regulator of growth and is known to be highly active in many types of human cancer. We find that mouse embryonic fibroblasts (MEFs) harboring constitutively active mTORC1 undergo apoptotic cell death when exposed to simultaneous deprivation of O2 and serum. Our recently published data demonstrate that apoptosis under these conditions occurs due to a limitation in unsaturated lipids and elevated ER-stress. This phenotype is not limited to MEFs; we find that a number of cancer cell lines also exhibit high levels of unsaturated lipid -dependent cell death under the same conditions, strongly suggesting that this mechanism of cell death is a widespread consequence of unregulated growth. Our data implicate the unfolded protein response (UPR) in mediating cell death and also suggest an important role for reactive oxygen species (ROS) in promoting apoptosis under these conditions. Our preliminary data suggest that the UPR sensor IRE1 and its downstream effector thioredoxin inhibiting protein (TXNIP) are critical in mediating cell death from unsaturated lipid deprivation. The central hypothesis of this proposal is that mTORC1 dysregulation under tumor-like stress induces apoptosis via a UPR- and ROS-dependent mechanism. Based on this hypothesis, I will pursue the following specific aims: Specific Aim 1: To determine how the UPR promotes cell death under tumor- like stress. Specific Aim 2: To test the involvement of TXNIP-mediated changes in redox status and metabolism in mediating cell death under tumor-like stress. Preliminary data using autochthonous Tsc2-/- renal tumors suggests an in vivo role for the cell death phenotype described here. We will compliment this model with an allograft tumor model and use it to test whether ER stress-mediated apoptosis can be exploited to target hypoxic tumor cells. To this end, I will pursue Specific Aim 3: To establish the in vivo relevance of mTORC1-driven cell death under nutrient and O2 deprivation. The studies proposed will help identify novel strategies for targeting cancer cells specifically.